U.S. patent application number 17/086031 was filed with the patent office on 2021-11-18 for protein transduction domain, fusion compound containing the same, and pharmaceutical composition containing the fusion compound.
The applicant listed for this patent is INDUSTRY ACADEMIC COOPERATION FOUNDATION, HALLYM UNIVERSITY. Invention is credited to Hyun Ju CHA, Soo-Young CHOI, Yeon Joo CHOI, Won Sik EUM, Kyuhyung HAN, Dae Won KIM, Hyun Jung KWON, Keunwook LEE, Sung Ho LEE, Sunghou LEE, Jin-Seu PARK, Jong Kook PARK, Soojung PARK, Min Jea SHIN, Eun Jeong SOHN, Eun Ji YEO, Hyeon Ji YEO.
Application Number | 20210355240 17/086031 |
Document ID | / |
Family ID | 1000005473414 |
Filed Date | 2021-11-18 |
United States Patent
Application |
20210355240 |
Kind Code |
A1 |
CHOI; Soo-Young ; et
al. |
November 18, 2021 |
PROTEIN TRANSDUCTION DOMAIN, FUSION COMPOUND CONTAINING THE SAME,
AND PHARMACEUTICAL COMPOSITION CONTAINING THE FUSION COMPOUND
Abstract
A protein transduction domain and a fusion compound, which are
more efficient, are proposed. As a result of selecting and testing
a number of candidate peptides, the present inventors found that a
GK1 peptide comprising 18 amino acids and a modified sequence
obtained by replacing, adding, or deleting some sequences are
bonded to high-molecular-weight materials such as proteins based on
the basic sequence of the peptide, thus enabling the
high-molecular-weight materials to smoothly penetrate into living
bodies, for example, cells, tissues, or blood. A fusion compound,
oligonucleotide, or vector using the same may be applied as a
pharmaceutical composition for preventing or treating diseases.
Inventors: |
CHOI; Soo-Young;
(Chuncheon-si, KR) ; PARK; Jin-Seu; (Chuncheon-si,
KR) ; HAN; Kyuhyung; (Chuncheon-si, KR) ; LEE;
Keunwook; (Seoul, KR) ; PARK; Jong Kook;
(Chuncheon-si, KR) ; LEE; Sunghou; (Yongin-si,
KR) ; LEE; Sung Ho; (Seoul, KR) ; PARK;
Soojung; (Incheon, KR) ; EUM; Won Sik;
(Chuncheon-si, KR) ; SHIN; Min Jea; (Incheon,
KR) ; YEO; Hyeon Ji; (Seoul, KR) ; YEO; Eun
Ji; (Seoul, KR) ; CHOI; Yeon Joo;
(Chungcheongbuk-do, KR) ; SOHN; Eun Jeong;
(Namyangju-si, KR) ; CHA; Hyun Ju;
(Dongducheon-si, KR) ; KWON; Hyun Jung;
(Gangneung-si, KR) ; KIM; Dae Won; (Gangneung-si,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY ACADEMIC COOPERATION FOUNDATION, HALLYM
UNIVERSITY |
Chuncheon-si |
|
KR |
|
|
Family ID: |
1000005473414 |
Appl. No.: |
17/086031 |
Filed: |
October 30, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/01 20130101;
A61K 8/64 20130101; C07K 19/00 20130101; A61K 38/00 20130101; C07K
14/4702 20130101 |
International
Class: |
C07K 19/00 20060101
C07K019/00; C07K 14/47 20060101 C07K014/47 |
Foreign Application Data
Date |
Code |
Application Number |
May 13, 2020 |
KR |
10-2020-0056941 |
Claims
1. A protein transduction domain which is any one selected from (A)
to (C) below, comprising 18 to 29 amino acids, and is chemically
bonded to a cargo molecule to thus transport the cargo molecule
into mammalian cell or tissue: TABLE-US-00004 (A)
R.sub.1R.sub.2R.sub.3DLAEGLAELADTR.sub.4R.sub.5R.sub.6, (R.sub.1 is
A or R, R.sub.2 is A or R, R.sub.3 is N or R, R.sub.4 is V or R,
R.sub.5 is G or R, and R.sub.6 is V or R), (B)
R.sub.7AANDLAEGLAELADTVGV or AANDLAEGLAELADTVGVR.sub.7, (R.sub.7 is
R, RR, or RRR), (C) R.sub.8AANDLAEGLAELADTVGV or
AANDLAEGLAELADTVGVR.sub.8, (R.sub.8 is RKKRRQRRR, KETWWET, or
EWSQPKKKRKV).
2. The protein transduction domain of claim 1, wherein the protein
transduction domain is any one selected from SEQ ID NO: 1 to SEQ ID
NO: 23.
3. The protein transduction domain of claim 1, wherein the cargo
molecule is selected from among proteins, peptides, amino acids,
nucleic acids, carbohydrates, lipids, and a mixture of one or more
thereof.
4. The protein transduction domain of claim 1, wherein bonding
between the cargo molecules and the protein transduction domain is
covalent bonding or non-covalent bonding.
5. A fusion compound penetrating into mammalian cell or tissue,
comprising: a protein transduction domain which is any one selected
from (A) to (C) below, comprising 18 to 29 amino acids, and is
chemically bonded to cargo molecule for preventing or treating
diseases to thus transport the cargo molecule into mammalian cell
or tissue: TABLE-US-00005 (A)
R.sub.1R.sub.2R.sub.3DLAEGLAELADTR.sub.4R.sub.5R.sub.6, (R.sub.1 is
A or R, R.sub.2 is A or R, R.sub.3 is N or R, R.sub.4 is V or R,
R.sub.5 is G or R, and R.sub.6 is V or R), (B)
R.sub.7AANDLAEGLAELADTVGV or AANDLAEGLAELADTVGVR.sub.7, (R.sub.7 is
R, RR, or RRR), (C) R.sub.8AANDLAEGLAELADTVGV or
AANDLAEGLAELADTVGVR.sub.8, (R.sub.8 is RKKRRQRRR, KETWWET, or
EWSQPKKKRKV).
6. The fusion compound of claim 5, wherein the protein transduction
domain is any one selected from SEQ ID NO: 1 to SEQ ID NO: 23.
7. The fusion compound of claim 5, wherein the cargo molecule is
selected from among proteins, peptides, amino acids, nucleic acids,
carbohydrates, lipids, and a mixture of one or more thereof.
8. A method for treating disease in a subject, said method
consisting of administering to said subject a therapeutically
effective amount of the fusion compound penetrating into mammalian
cell or tissue of claim 5.
9. A cosmetic composition comprising: the fusion compound
penetrating into mammalian cell or tissue of claim 5.
10. A recombinant polynucleotide encoding a fusion compound
penetrating into mammalian cell or tissue, in which an
oligonucleotide sequence encoding the protein transduction domain
of claim 1 is chemically bonded to a cDNA sequence encoding cargo
molecule for preventing or treating diseases so that the protein
transduction domain is chemically bonded to the cargo molecule for
preventing or treating diseases.
11. The recombinant polynucleotide of claim 10, wherein the
oligonucleotide sequence encoding the protein transduction domain
is any one selected from SEQ ID NO: 24 to SEQ ID NO: 46.
12. A pharmaceutical composition for preventing or treating
diseases, comprising: the recombinant polynucleotide encoding a
fusion compound of claim 10 penetrating into mammalian cell or
tissue.
13. A fusion compound expression vector comprising: the recombinant
polynucleotide encoding a fusion compound of claim 10 penetrating
into mammalian cell or tissue.
14. A pharmaceutical composition for preventing or treating
diseases, comprising: the fusion compound expression vector of
claim 13.
Description
RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
USC.sctn. 119(d) of Korean Patent Application No. 10-2020-0056941,
filed May 13, 2020, the content of which is incorporated by
reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled 34468813_1. TXT, created and last modified on Feb.
23, 2021, which is 16.5 Kb in size. The information in the
electronic format of the Sequence Listing is incorporated herein by
reference in its entirety.
BACKGROUND OF THE DISCLOSURE
1. Field of the Disclosure
[0003] The present disclosure relates to a protein transduction
domain that comprises a peptide of the 18 amino acids
AANDLAEGLAELADTVGV or a peptide obtained by deleting, replacing, or
adding some amino acids thereof. Further, the present disclosure
relates to a fusion compound in which the protein transduction
domain is bonded to cargo molecules, and to a pharmaceutical
composition including the fusion compound.
2. Description of the Related Technology
[0004] A protein transduction domain (PTD) or cell-penetrating
peptide (CPP) is a delivery system that fuses a peptide comprising
approximately 5 to 30 amino acids with high-molecular-weight
materials such as proteins or nucleic acids, thus easily delivers
the fused substances into living bodies, for example, mammalian
cells, tissues, or blood. Although the specific mechanism has not
yet been accurately identified, a protein delivery technology has
been used to deliver proteins into cells or tissues for in-vitro or
in-vivo treatment, and various protein transduction domains are
known. The protein transduction domain is conveniently fused with
biological cargo molecules for transduction and does not affect the
structure or function of the cargo molecules, and thus the fused
compound is stable. Accordingly, the protein transduction domain is
used in medicines and cosmetics.
[0005] The disclosure of this section is to provide background
information relating to the invention. Applicant does not admit
that any information contained in this section constitutes prior
art.
SUMMARY OF THE DISCLOSURE
[0006] Therefore, an aspect of the present disclosure provides a
novel protein transduction domain that is more efficient and safer
than a conventional protein transduction domain.
[0007] Another aspect of the present disclosure is to provide a
fusion compound bonded to the novel protein transduction
domain.
[0008] Another aspect of the present disclosure is to provide a
pharmaceutical composition including the fusion compound.
[0009] Another aspect of the present disclosure is to provide a
cosmetic composition including the fusion compound.
[0010] Another aspect of the present disclosure is to provide a
method for smoothly transporting useful high-molecular-weight
materials such as proteins or nucleic acids into cells or
tissues.
[0011] As a result of selecting and testing a number of candidate
peptides, the present inventors found that a peptide comprising the
18 amino acids AANDLAEGLAELADTVGV (SEQ ID NO: 1) (hereinafter,
referred to as "GK1" in the present disclosure) or a peptide
obtained by deleting, replacing, or adding some amino acids thereof
enables high-molecular-weight materials such as proteins to
smoothly transport into living bodies, for example, cells, tissues,
or blood.
[0012] Definitions of main terms used in the description and claims
of the present disclosure are as follows.
[0013] "Cargo molecules" are molecules other than a protein
transduction domain or fragments thereof, which cannot enter target
cells naturally or cannot enter target cells at the useful rate
naturally. "Cargo molecules" refer to the target molecules before
fusion with the protein transduction domain or the target molecule
part of the protein transduction domain-target molecule complex.
The cargo molecules refer to any one selected from among proteins,
peptides, amino acids, nucleic acids, carbohydrates, lipids, and a
mixture of one or more thereof.
[0014] "Target proteins" as a concept included in the "cargo
molecules" are molecules other than a protein transduction domain
or fragments thereof, which cannot enter target cells naturally or
cannot enter target cells at the useful rate naturally. The target
proteins refer to the target protein molecules before fusion with
the protein transduction domain or the target protein part of the
protein transduction domain-target protein complex. The target
proteins include polypeptide, protein, or peptide. Examples of the
target proteins may include PGAM1 (phosphoglycerate mutase 1),
BLVRA (biliverdin reductase A), superoxide dismutase, an epithelial
cell growth factor, a fibroblast growth factor, catalase, and
FK506BP (FK506-binding protein). However, it is obvious to a person
skilled in the art that the above examples are only partial
illustrations of the target protein, and the target protein is not
limited thereto.
[0015] "Fusion proteins" include a transduction domain and one or
more target protein parts, and the term refers to a complex formed
by genetic fusion or chemical bonding between the transduction
domain and the target protein.
[0016] Further, "genetic fusion" refers to a linear linkage
obtained by covalent bonding through the genetic expression of a
DNA sequence encoding a protein. Further, "target cells" means
cells to which a target protein is delivered by a protein
transduction domain. The target cells refer to intracorporeal or
extracorporeal cells. In other words, "target cells" is meant to
include intracorporeal cells, that is, cells that make up organs or
tissues of living animal or human, or microorganisms found in
living animals or humans. Further, "target cells" is meant to
include extracorporeal cells, that is, cultured animal cells,
cultured human cells, or cultured microorganisms.
[0017] "Protein transduction domain" in the present disclosure
refers to a peptide that forms a covalent bond with
high-molecular-weight organic compounds, for example,
oligonucleotides, peptides, proteins, oligosaccharides, or
polysaccharides, thereby introducing the organic compounds into
cells or tissues without requiring separate receptors, carriers, or
energy.
[0018] Further, in this specification, with respect to the
"transduction" of proteins, peptides, or organic compounds into
cells or tissues, the terms "penetrating" and "transporting" may be
used interchangeably therewith.
[0019] The present disclosure relates to a protein transduction
domain which is any one selected from (A) to (C) below, includes 18
to 29 amino acids, and is chemically bonded to cargo molecules to
thus transport the cargo molecules into mammalian cells or
tissues.
[0020] (A) R1R2R3DLAEGLAELADTR4R5R6 (SEQ ID NO: 49; R1 is A or R,
R2 is A or R, R3 is N or R, R4 is V or R, R5 is G or R, and R6 is V
or R),
[0021] (B) R7-AANDLAEGLAELADTVGV (SEQ ID NO: 50) or
AANDLAEGLAELADTVGV-R7 (SEQ ID NO: 51) (R7 is R, RR, or RRR),
[0022] (C) R8-AANDLAEGLAELADTVGV (SEQ ID NO: 52) or
AANDLAEGLAELADTVGV-R8 (SEQ ID NO: 53) (R8 is RKKRRQRRR (SEQ ID NO:
54), KETWWET (SEQ ID NO; 55), or EWSQPKKKRKV (SEQ ID NO: 56)).
[0023] Further, in the present disclosure, the protein transduction
domain is any one selected from SEQ ID NO: 1 to SEQ ID NO: 23. The
protein transduction domain of the present disclosure is not
limited to SEQ ID NO: 1 to SEQ ID NO: 23, but it should be noted
that representative peptides are listed in Table 1 for convenience
of experimentation.
[0024] "Protein transduction domain" according to the present
disclosure is interpreted to mean including variants, in which one
or more amino-acid residues are conservatively substituted at
specific amino-acid residue positions, or fragments thereof
including eighteen or more amino acids among fragments in which one
to three amino acids are deleted at the N-terminus and/or
C-terminus thereof.
[0025] In the present specification, "conservative substitution"
refers to modification of a protein transduction domain, including
substitution of one or more amino acids with amino acids that do
not cause loss of biological or biochemical functions of the
corresponding protein transduction domain and have biochemical
properties similar thereto.
[0026] In the present specification, "conservative substitution of
amino acids" is a substitution of amino acid residues with amino
acid residues having similar side chains. Classes of amino acid
residues having similar side chains can be defined. Examples
thereof include amino acids having basic side chains (for example,
lysine, arginine, and histidine), amino acids having acidic side
chains (for example, aspartic acid and glutamic acid), amino acids
having uncharged polar side chains (for example, glycine,
asparagine, glutamine, serine, threonine, tyrosine, and cysteine),
amino acids having non-polar side chains (for example, alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
and tryptophan), amino acids having beta-branched side chains (for
example, threonine, valine, and isoleucine), and amino acids having
aromatic side chains (for example, tyrosine, phenylalanine,
tryptophan, and histidine).
[0027] It is expected that the activity of the protein transduction
domain of the present disclosure or fragments thereof will be
maintained even when conservative substitution of amino acids is
performed.
[0028] Further, the protein transduction domain variant according
to the present disclosure is interpreted to have substantially the
same function and/or effect as the protein transduction domain
according to the present disclosure, and has the meaning of
including protein transduction domain variants or fragments thereof
having an amino acid sequence homology of 80% or 85% or more, in an
embodiment, 90% or more, and in another embodiment, 95% or
more.
[0029] Further, in the present disclosure, the cargo molecules are
selected from among proteins, peptides, amino acids, nucleic acids,
carbohydrates, lipids, and a mixture of one or more thereof.
[0030] Further, in the present disclosure, the cargo molecules are
selected from among nanoparticles, microparticles, liposomes, and
micelles.
[0031] Further, in the present disclosure, the chemical bonding of
the protein transduction domain and the cargo molecules is covalent
bonding or non-covalent bonding. The chemical bonding may be
covalent bonding or non-covalent bonding. Examples of the
non-covalent bonding may include ionic bonding, bonding by
electrostatic attraction, or bonding by hydrophobic interaction.
Further, the material capable of being bonded to the protein
transduction domain by the ionic bonding or electrostatic
attraction may be a material having one or more charges, such as
DNA or RNA.
[0032] Further, the present disclosure relates to a fusion compound
that easily penetrates into cells or tissues. The fusion compound
includes a protein transduction domain which is any one selected
from (A) to (C) as described above and is bonded to cargo molecules
for preventing or treating diseases to transport the cargo
molecules into cells or tissues.
[0033] Further, the present disclosure relates to a fusion compound
that easily penetrates into cells or tissues, in which the protein
transduction domain is any one selected from SEQ ID NO: 1 to SEQ ID
NO: 23. The protein transduction domain of the present disclosure
is not limited to SEQ ID NO: 1 to SEQ ID NO: 23, but it should be
noted that representative peptides are listed in Table 1 for
convenience of experimentation.
[0034] Further, the present disclosure relates to a fusion compound
in which the cargo molecules are selected from among proteins,
peptides, amino acids, nucleic acids, carbohydrates, lipids, and a
mixture of one or more thereof.
[0035] Further, the present disclosure relates to a fusion compound
in which the cargo molecules are selected from among nanoparticles,
microparticles, liposomes, and micelles.
[0036] Further, the present disclosure relates to a pharmaceutical
composition for preventing or treating diseases including the
above-described fusion compound, which easily penetrates into cells
or tissues.
[0037] Further, the present disclosure relates to a cosmetic
composition including the above-described fusion compound, which is
easily transduced into cells or tissues. The cosmetic composition
of the present disclosure may include basic cosmetics, such as
lotions, creams, essences, oil-in-water- or water-in-oil-type of
emulsions, and ointments, as well as color cosmetics such as
foundation, lipstick, and eye shadow.
[0038] Further, the present disclosure relates to a recombinant
polynucleotide encoding a fusion compound that easily penetrates
into cells or tissues, in which an oligonucleotide sequence
encoding the protein transduction domain is bonded to a cDNA
sequence encoding cargo molecules for preventing or treating
diseases so that the protein transduction domain is chemically
bonded to the cargo molecules for preventing or treating
diseases.
[0039] Further, the present disclosure relates to a recombinant
polynucleotide encoding a fusion compound that easily penetrates
into cells or tissues, in which the oligonucleotide sequence
encoding the protein transduction domain is any one selected from
SEQ ID NO: 24 to SEQ ID NO: 46. The oligonucleotide encoding the
protein transduction domain of the present disclosure is not
limited to SEQ ID NO: 24 to SEQ ID NO: 46, but it should be noted
that representative oligonucleotides are illustrated in Table 2 for
convenience of experimentation.
[0040] Further, the present disclosure relates to a pharmaceutical
composition for preventing or treating diseases, which includes the
recombinant polynucleotide encoding a fusion compound that easily
penetrates into cells or tissues.
[0041] Further, the present disclosure relates to a method for
treating disease in a subject, said method consisting of
administering to said subject a therapeutically effective amount of
the fusion compound penetrating into mammalian cell or tissues. The
fusion protein of the present disclosure invention can be
administered to a subject suffering from such a disease or
suspected of a disease to prevent or treat a disease caused by a
lack of a cargo protein.
[0042] Further, the present disclosure relates to a fusion-compound
expression vector including the recombinant polynucleotide encoding
a fusion compound that easily penetrates into cells or tissues.
[0043] Further, the present disclosure relates to a pharmaceutical
composition for preventing or treating diseases, which includes the
fusion-compound expression vector.
[0044] A pharmaceutical composition that includes the fusion
compound of the present disclosure, an oligonucleotide encoding the
same, or a vector containing the oligonucleotide as an active
ingredient may be blended with a typical carrier acceptable for use
in the pharmaceutical field, and may be formulated in various
forms, such as external skin preparations, oral types, spray types,
patches, or injection types, according to a typical method. For
example, the oral compositions include tablets and gelatin
capsules. The oral compositions include, in addition to the active
ingredients, a diluent (e.g.: lactose, dextrose, sucrose, mannitol,
sorbitol, cellulose, and/or glycine), and a lubricating agent
(e.g.: silica, talc, stearic acid and magnesium or calcium salts
thereof, and/or polyethylene glycol). In embodiments, tablet
includes a binding agent (e.g.: magnesium aluminum silicate, starch
paste, gelatin, methylcellulose, sodium carboxymethylcellulose,
and/or polyvinyl pyrrolidone), and includes a disintegrant (e.g.:
starch, agar, alginic acid or sodium salts thereof), a boiling
mixture and/or an absorbent, a colorant, a flavoring agent, and a
sweetening agent, in some cases. In embodiments, the composition
for injection is an isotonic aqueous solution or suspension. The
above-described composition is sterilized and/or contains an
adjuvant (e.g.: a preservative, a stabilizer, a wetting- or
emulsification-promoting agent, and salts and/or buffers for
controlling osmotic pressure). Further, other materials that are
therapeutically useful may be contained therein.
[0045] The pharmaceutical preparation prepared as described above
may be orally administered, may be parenterally administered, that
is, intravenously, subcutaneously, or intraperitoneally
administered, or may be topically applied depending on the purpose
thereof. With respect to the dose thereof, the pharmaceutical
preparation may be administered by dividing the daily dose of
0.0001 to 100 mg/kg into one to several administrations. The dosage
level for a specific patient may depend on the patient's weight,
age, sex, and health status, administration time, administration
method, excretion rate, and severity of disease.
[0046] In one embodiment in the present disclosure provides a
method for treating disease in a subject, said method consisting of
administering to said subject a therapeutically effective amount of
the fusion compound penetrating into mammalian cell or tissues. In
another embodiment in the present disclosure provides a method for
treating disease in a subject, said method comprising administering
to said subject a therapeutically effective amount of the fusion
compound penetrating into mammalian cell or tissues. The fusion
protein according to embodiments of the present invention can be
administered to a subject suffering from such a disease or
suspected of a disease to prevent or treat a disease caused by a
lack of a cargo protein.
[0047] The novel protein transduction domain of the present
disclosure may be bonded to cargo molecules such as proteins to
form a fusion compound, thereby easily penetrating into cells or
tissues.
[0048] Further, the fusion compound, in which the novel protein
transduction domain of the present disclosure is bonded to cargo
molecules, exhibits activity in cells or tissues. Accordingly, the
fusion compound, the oligonucleotide encoding the same, or a vector
including the oligonucleotide may be used as a pharmaceutical
composition for preventing or treating diseases.
[0049] Further, it is possible to enable cargo molecules such as
proteins to easily penetrate into cells or tissues using the novel
protein transduction domain of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The above and other aspects, features and advantages of the
present disclosure will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0051] FIGS. 1A and 1B show the construction of a GK1 expression
vector on a pET-15b vector. A synthetic GK1 oligomer was cloned at
the NdeI site, and human PGAM1 (phosphoglycerate mutase 1) cDNA or
human BLVRA (biliverdin reductase A) cDNA was cloned at the XhoI
and BamHI sites of pET-15b.
[0052] FIG. 2 shows results of western blotting analysis. A of FIG.
2 shows the result of western blotting analysis of the level of
cell-transduced proteins after cells are treated with a Pep-1-PGAM1
fusion protein, a GK1-PGAM1 fusion protein, or a Tat-PGAM1 fusion
protein for 1 hour. B of FIG. 2 shows the results of western
blotting of cells treated with a GK1-PGAM1 fusion protein or a
GK1-BLVRA fusion protein.
[0053] FIG. 3A shows the intracellular locations and levels of a
cell-transduced control PGAM1 protein, a Pep-1-PGAM1 fusion
protein, a GK1-PGAM1 fusion protein, and a Tat-PGAM1 fusion
protein, which are photographed using a confocal fluorescence
microscope, and FIG. 3B shows the intracellular locations and
levels of a cell-transduced control PGAM1 protein, a GK1-PGAM1
fusion protein, a control BLVRA protein, and a GK1-BLVRA fusion
protein, which are photographed using a confocal fluorescence
microscope.
[0054] FIG. 4 is a graph showing the protective effect of each
protein against oxidative stress. Cells were pre-treated with each
of a control PGAM1 protein, a GK1-PGAM1 fusion protein, a control
BLVRA protein, and a GK1-BLVRA fusion protein at a concentration of
5 .mu.M for 1 hour, and were then treated with 100 .mu.M
H.sub.2O.sub.2 for 1 hour. Then, cell viability was evaluated using
an MTT analysis method.
[0055] FIG. 5A and FIG. 5B show the intracellular location and
level of a cell-transduced PGAM1 fusion protein, which are
photographed using a confocal fluorescence microscope. The median
number of the fusion protein is the name of the protein
transduction domain shown in Table 1.
[0056] FIG. 6 shows the results obtained by testing the protective
effect of cell-transduced PGAM1 fusion proteins against oxidative
stress. Cells were treated with GK1-PGAM1 fusion proteins and a
control PGAM1 protein at a concentration of 5 .mu.M for 1 hour, and
were then treated with 100 .mu.M H.sub.2O.sub.2 for 1 hour. Then,
cell viability was evaluated using an MTT analysis method. The
median number of the fusion protein is the number of the protein
transduction domain shown in Table 1.
DESCRIPTION OF EMBODIMENTS
[0057] Hereinafter, the configuration of the present disclosure
will be described in more detail with reference to specific
Examples. However, it is apparent to those of ordinary skill in the
art that the scope of the present disclosure is not limited only to
the description of the Examples.
[0058] The numerous protein transduction domains may be divided
into three categories. The first is an amphiphilic peptide, and
examples thereof may include a Pep-1 peptide. The Pep-1 peptide
consists of 21 amino acids (KETWWETWWTEWSQPKKKRKV) and has three
domains, namely a hydrophobic domain, a spacer, and a hydrophilic
domain. The second is a cationic peptide, and examples thereof may
include a HIV-Tat peptide (Hereinafter, "Tat" or "Tat peptide"),
oligolysine, oligoarginine, and oligo (lysine+arginine). The
sequence of the cell-transducing Tat peptide includes RKKRRQRRR,
and the peptide has been used in the study of various proteins for
treatment. The third is a hydrophobic peptide.
[0059] A bonding between a protein transduction domain and a
biological cargo molecule (for example, nucleic acids, proteins,
peptides, small molecules, cytotoxic drugs etc.) may be
accomplished using various methods such as ionic bond and
electrostatic bond, in addition to covalent bond.
[0060] The protein transduction domain has advantages of exhibiting
lower toxicity and less frequent immunorejection than other
delivery substances such as liposomes or polymers. However, few
protein transduction domains are used in clinical practice.
[0061] <Method>
[0062] 1. Cell Culture
[0063] NSC34 cell line, which is a mouse-motoneuron-like hybrid
cell line, was distributed from College of Natural Sciences of
Hallym University. The cell culture solution was prepared by adding
10% fetal bovine serum (FBS) and an antibiotic solution (100
units/ml penicillin, 100 .mu.g/ml streptomycin) to a DMEM medium.
The NSC34 cell line was cultured using the above cell culture
solution at 37.degree. C. and 95% humidity under the condition in
which 5% CO.sub.2 was maintained. When the cell reaches 70 to 80%
confluent, treatment with trypsin-EDTA was performed to accomplish
subculture.
[0064] 2. Construction of Recombinant GK1
[0065] Annealing was performed at 37.degree. C. for 2 hours using
the following oligonucleotides.
TABLE-US-00001 Sense oligonucleotide (SEQ ID NO: 47):
tatggctgcgaatgatcttgcggaaggtctggcggaactcgcggatacgg taggcgtaca,
Anti-sense oligonucleotide (SEQ ID NO: 48):
tatgtacgcctaccgtatccgcgagttccgccagaccttccgcaagatca ttcgcagcca.
[0066] Thereafter, the NdeI site of a pET-15b vector was cut, and
the GK1-coding oligonucleotide pairs prepared in the above were
connected. Transformation of the Escherichia coli strain Top 10
with the recombinant GK1 was performed. The DNA separated from the
transformed cell was amplified through PCR using the following
primers.
[0067] Forward primer: T7, reverse primer: T7 terminator.
[0068] The BamHI and XhoI sites of the GK1 vector were cut to
connect PGAM1 (phosphoglycerate mutase 1), BLVRA (biliverdin
reductase A) or CNRIP1 (Cannabinoid Receptor Interacting Protein 1)
gene.
[0069] 3. Expression and Purification of Recombinant GK1
[0070] Transformation of Escherichia coli strain BL21 with the
recombinant GK1 plasmid was performed. After the transformed cell
was cultured in 100 ml LB medium containing 100 .mu.g/ml ampicillin
at 37.degree. C. and 180 rpm for 6 hours, 0.5 mM IPTG
(isopropyl-.beta.-D-thiogalactoside) was added thereto, and then
culture was performed at 37.degree. C. and 120 rpm for 16 hours.
The recovered cells were subjected to ultrasonic treatment and
centrifuged to isolate only a supernatant, followed by purification
using an Ni.sup.2+-nitrilotriacetic acid Sepharose affinity
chromatography column. A protein concentration was normalized with
bovine serum albumin (BSA) and quantified using a protein
quantitative assay kit.
[0071] 4. Transduction of GK1 Fusion Protein into NSC34 Cells
[0072] The NSC34 cell was dispensed on a 60 mm plate and cultured
under conditions of 37.degree. C., 95% humidity, and 5% CO.sub.2.
In order to examine the cell penetration capability of GK1,
Pep-1-PGAM1 fusion protein, Tat-PGAM1 fusion protein, and GK1-PGAM1
fusion protein at the same concentration (5 .mu.M) were used in
treatment for 20 minutes, respectively. Further, in order to
confirm that the cell penetration capability of the protein
transduction domain GK1 is shown in arbitrary protein, another
protein at the same concentration, namely, a GK1-BLVRA fusion
protein, was used in treatment for 20 minutes. The cultured cells
were rinsed with PBS (phosphate-buffered saline). In order to
extract proteins, a RIPA lysis buffer was added thereto and
centrifuged (4.degree. C., 12,000 rpm, and 10 min), thereby
performing protein quantification with respect to the
supernatant.
[0073] In order to compare the cell transduction efficiency, a
Pep-1-PGAM1 fusion protein and a Tat-PGAM1 fusion protein that are
fusion proteins using Pep-1 peptide and Tat peptide, were prepared
according to a method similar to the method described above (Kim W.
et al., Neurochemistry International, 10 Dec. 2019, 133:104631).
Thereafter, the Pep-1-PGAM1 fusion protein and the Tat-PGAM1 fusion
protein were transduced into NSC34 cell according to the method as
described above.
[0074] 5. Western Blotting Analysis
[0075] 50 .mu.g of the fusion protein or the control protein was
mixed with a 5.times. sample buffer and then boiled for 2 minutes
to prepare a protein sample, followed by separation according to
molecular weight using 15% mini gel SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel electrophoresis). After the
electrophoresis was finished, the resultant was transferred to a
nitrocellulose membrane, and blocking of the membrane was carried
out using TBS-T (pH 7.5, 25 mM Tris-Cl, 150 mM NaCl, and 0.1% Tween
20) containing 5% skim milk at room temperature for 1 hour. In
order to measure the expression of the protein, a primary antibody
(anti-histidine probe) was diluted in the TBS-T at a ratio of
1:1,000, reacted at room temperature for 1 hour, and then washed
with the TBS-T. A HRP (horseradish
peroxidase)-conjugated-anti-rabbit IgG as a secondary antibody was
diluted in TBS-T at a ratio of 1:10,000 to be reacted at room
temperature for 1 hour. After washing with the TBS-T, the
expression level of each protein was confirmed using a detection
reagent.
[0076] 6. Fluorescence Microscope Analysis
[0077] Each glass cover slip was placed on a 24-well plate, and
cells were dispensed thereon and cultured for 24 hours. After 5
.mu.M of the Pep-1-PGAM1 fusion protein, Tat-PGAM1 fusion protein,
GK1-PGAM1 fusion protein, or GK1-BLVRA fusion protein was
transduced into an NSC34 cell for 2 hours, the culture medium was
removed, and rinsing was repeated with PBS three times. After 4%
para-formaldehyde was added to each well and the cells were fixed
for 5 minutes, PBS was added thereto, and washing was repeated
three times for a short time. After blocking and penetration in PBS
(PBS-BT) containing 3% BSA and 0.1% Triton X-100 at room
temperature for 40 minutes, washing with PBS-BT was repeated three
times. A His-probe primary antibody was diluted at a ratio of
1:2,000 and cultured at room temperature for 1 hour. A secondary
antibody (Alexa Fluor 488) was diluted to a ratio of 1:10,000,
reacted for 1 hour in dark, and washed with the PBS-BT three times
for 5 minutes. The glass cover slip was separated from the well to
remove moisture from the edge thereof, and was lifted. A
fluorescence microscope was used for observation, and fluorescence
emission was confirmed using an image analyzer.
[0078] <Result>
[0079] Result 1: Schematic Diagram and Purification of GK1 Fusion
Protein
[0080] In order to express the protein transduction domain, cloning
of a GK1 oligonucleotide (gct gcg aat gat ctt gcg gaa ggt ctg gcg
gaa ctc gcg gat acg gta ggc gta; SEQ ID NO: 24) was performed at an
NdeI site of a pET-15b plasmid. In order to confirm the cell
penetration efficiency of the protein transduction domain GK1,
cloning of PGAM1 (phosphoglycerate mutase 1) cDNA or BLVRA
(biliverdin Reductase A) cDNA was performed at XhoI and BamHI sites
of the pET-15b plasmid containing GK1 oligonucleotide. In the case
of a control vector, cloning of the PGAM1 protein or the BLVRA
protein was performed at the pET-15b plasmid not containing the GK1
oligonucleotide. After expression of the fusion protein or the
protein, purification was performed using an
Ni.sup.2+-nitrilotriacetic acid Sepharose affinity chromatography
column, and was confirmed using SDS-PAGE [FIGS. 1A and 1B].
[0081] Result 2: Transduction of GK1 Fusion Protein into Cell
[0082] In order to examine the cell transduction efficiency of GK1,
the Pep-1-PGAM1 fusion protein, the GK1-PGAM1 fusion protein, or
the Tat-PGAM1 fusion protein at the same concentration (5 .mu.M)
was used to treat an NSC34 cell for 1 hour. Then, the cell
transduction level was analyzed by western blotting. The level of
intracellular fusion protein was highest in the case of the
GK1-PGAM1 fusion protein. In comparison with the GK1-PGAM1 fusion
protein, the Tat-PGAM1 fusion protein showed a cell transduction
level of about 89%, and the Pep-1-PGAM1 fusion protein showed a
cell transduction level of about 50% [A of FIG. 2]. The control
PGAM1 protein did not penetrate into the cells.
[0083] In order to prove that the cell-penetrating domain GK1 was
fused with an arbitrary protein to penetrate the cell, the
GK1-PGAM1 fusion protein and the GK1-BLVRA fusion protein, prepared
by fusing the protein transducing domain GK1 with the two types of
proteins PGAM1 and BLVRA respectively were used at the same
concentration (5 .mu.M) to treat the cell for 1 hour. Although
there is a difference in cell transduction efficiency depending on
the protein type, the two types of proteins smoothly penetrated
into the NSC34 cells [B of FIG. 2].
[0084] In order to examine the location of the Pep-1-PGAM1 fusion
protein, GK1-PGAM1 fusion protein, or Tat-PGAM1 fusion protein
transduced into the cells, the cells into which each fusion protein
penetrated were subjected to immunostaining using DAPI and Alexa
Fluor 488-conjugated secondary antibodies. A control PGAM1 protein
was not observed in the NSC34 cell. Unlike this, the Pep-1-PGAM1
fusion protein, the GK1-PGAM1 fusion protein, and the Tat-PGAM1
fusion protein were detected mainly in the cytoplasm using a
fluorescence microscope, and were also detected in the nucleus
[FIG. 3A]. Further, in the case of the GK1-PGAM1 fusion protein, a
fluorescence signal having a higher intensity was observed compared
to the Pep-1-PGAM1 fusion protein and the Tat-PGAM1 fusion protein.
This implies that the cell transduction efficiency of the GK1-PGAM1
fusion protein is the highest.
[0085] Further, the intracellular location of the GK1 fused with an
arbitrary protein to penetrate into a cell was examined using the
above-described method. The GK1-PGAM1 fusion protein and the
GK1-BLVRA fusion protein were detected mainly in the cytoplasm, and
were also detected in the nucleus [FIGS. 3A and 3B].
[0086] Result 3: Effect of GK1 Fusion Protein Against Oxidative
Stress
[0087] It was examined whether the GK1 fusion protein transducing
into cells had a protective effect against oxidative stress.
Treatment was performed with a GK1-PGAM1 fusion protein, a
GK1-CNRIP1 fusion protein, a control PGAM1 protein, and a control
CNRIP1 protein at a constant concentration (5 .mu.M) for 1 hour,
followed by treatment with 100 .mu.M H.sub.2O.sub.2 for 1 hour.
Thereafter, cell viability was evaluated using an MTT analysis
method [FIGS. 5A and 5B]. The viability of the cell treated with
H.sub.2O.sub.2 was reduced by about 57% compared to the control
cell. The viability of the cell pre-treated with GK1-PGAM1 fusion
protein or GK1-BLVRA fusion protein was increased by about 83%. In
comparison, the control PGAM1 protein and the control CNRIP1
protein had no protective effect against cell death caused by
H.sub.2O.sub.2.
[0088] Result 4: Transduction of Fusion Protein Using the GK1
Protein Transduction Domain into Cells and Effect Thereof Against
Oxidative Stress
[0089] A GK1 peptide sequence (AANDLAEGLAELADTVGV; SEQ ID NO: 1) or
a GK1 derivative peptide in which one or more peptides are
substituted into or added to a part of the sequence, sequences in
which partial consecutive sequence of the GK1 peptide and sequence
of Tat peptide or partial consecutive sequence thereof are bonded,
and sequences in which partial consecutive sequence of the GK1
peptide and native or partial consecutive sequence of the Tat
peptide, and native or partial consecutive sequence of the Pep-1
peptide are bonded are shown in Table 1. It should be noted that
the sequences listed in Table 1 are just some examples of the
protein transduction domain claimed in the present disclosure, and
the protein transduction domain of the present disclosure is not
limited to the sequences listed in Table 1.
[0090] In order to confirm the intracellular locations of the
cell-transduced fusion protein in which a GK1 derivative peptide
fused with the PGAM1 protein, immunostaining was performed in the
same manner as described above. The GK1 derivative peptides were
also detected in the cytoplasm and nucleus. Among them, eight
peptides having favorable cell transduction efficiency were
selected, and are shown in FIGS. 5A and 5B.
[0091] In order to examine the cell protection effect of the fusion
protein fused with the GK1 derivative peptide against oxidative
stress, MTT analysis was performed in the same manner as described
above. The cell viability, which had been reduced to 57% due to
H.sub.2O.sub.2, was increased to the minimum value of 56% to the
maximum value of 83% when pre-treatment was performed with the
fusion protein in which the GK1 candidate protein was fused with
the PGAM1 protein [FIG. 6]. In the case of the GK1-PGAM1 fusion
protein, the cell viability was increased by 80%.
[0092] Table 1 below shows twenty-three specific examples of amino
acid sequences of the protein transduction domain of the present
disclosure. Further, Table 2 below shows nucleotide sequences
encoding the twenty-three specific examples of the protein
transduction domain of the present disclosure.
TABLE-US-00002 TABLE 1 Seq. Name No. Peptide sequence GK1 1
AANDLAEGLAELADTVGV GK1-1 2 RANDLAEGLAELADTVGV GK1-2 3
RRNDLAEGLAELADTVGV GK1-3 4 RRRDLAEGLAELADTVGV GK1-4 5
AANDLAEGLAELADTVGR GK1-5 6 AANDLAEGLAELADTVRR GK1-6 7
AANDLAEGLAELADTRRR GK1-7 8 ARNDLAEGLAELADTVGV GK1-8 9
AARDLAEGLAELADTVGV GK1-9 10 AANDLAEGLAELADTVRV GK1-10 11
AANDLAEGLAELADTRGV GK1-11 12 RAANDLAEGLAELADTVGV GK1-12 13
RRAANDLAEGLAELADTVGV GK1-13 14 RRRAANDLAEGLAELADTVGV GK1-14 15
AANDLAEGLAELADTVGVR GK1-15 16 AANDLAEGLAELADTVGVRR GK1-16 17
AANDLAEGLAELADTVGVRRR GK1-17 18 RKKRRQRRRAANDLAEGLAELADTVGV GK1-18
19 AANDLAEGLAELADTVGVRKKRRQRRR GK1-19 20 KETWWETAANDLAEGLAELADTVGV
GK1-20 21 EWSQPKKKRKVAANDLAEGLAELADTVGV GK1-21 22
AANDLAEGLAELADTVGVKETWWET GK1-22 23
AANDLAEGLAELADTVGVEWSQPKKKRKV
TABLE-US-00003 TABLE 2 Seq. Name No. Nucleotide sequence GK1 24 gct
gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta
GK1-1 25 cgc gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat
acg gta ggc gta GK1-2 26 cgc cgt aat gat ctt gcg gaa ggt ctg gcg
gaa ctc gcg gat acg gta ggc gta GK1-3 27 cgc cgt cgc gat ctt gcg
gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta GK1-4 28 gct gcg
aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc cgc
GK1-5 29 gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat
acg gta cgc cgc GK1-6 30 gct gcg aat gat ctt gcg gaa ggt ctg gcg
gaa ctc gcg gat acg cgt cgc cgc GK1-7 31 gct cgc aat gat ctt gcg
gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta GK1-8 32 gct gcg
cgc gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta
GK1-9 33 gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat
acg gta cgc gta GK1-10 34 gct gcg aat gat ctt gcg gaa ggt ctg gcg
gaa ctc gcg gat acg cgc ggc gta GK1-11 35 cgc gct gcg aat gat ctt
gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta GK1-12 36 cgc
cgt gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta
ggc gta GK1-13 37 cgc cgt cgc gct gcg aat gat ctt gcg gaa ggt ctg
gcg gaa ctc gcg gat acg gta ggc gta GK1-14 38 gct gcg aat gat ctt
gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta cgc GK1-15 39
gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc
gta cgc cgt GK1-16 40 gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa
ctc gcg gat acg gta ggc gta cgc cgt cgc GK1-17 41 agg aag aag agg
aga cag cga cga aga gct gcg aat gat ctt gcg gaa ggt ctg gcg gaa ctc
gcg gat acg gta ggc gta GK1-18 42 gct gcg aat gat ctt gcg gaa ggt
ctg gcg gaa ctc gcg gat acg gta ggc gta agg aag aag agg aga cag cga
cga aga GK1-19 43 aaa gaa acc tgg tgg gaa acc gct gcg aat gat ctt
gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta GK1-20 44 gaa
tgg tct cag ccg aaa aaa aaa cgt aaa gtg gct gcg aat gat ctt gcg gaa
ggt ctg gcg gaa ctc gcg gat acg gta ggc gta GK1-21 45 gct gcg aat
gat ctt gcg gaa ggt ctg gcg gaa ctc gcg gat acg gta ggc gta aaa gaa
acc tgg tgg gaa acc GK1-22 46 gct gcg aat gat ctt gcg gaa ggt ctg
gcg gaa ctc gcg gat acg gta ggc gta gaa tgg tct cag ccg aaa aaa aaa
cgt aaa gtg
[0093] Although embodiments of the present disclosure have been
disclosed for illustrative purposes, those skilled in the art will
appreciate that various modifications, additions and substitutions
are possible, without departing from the scope and spirit of the
disclosure as disclosed in the accompanying claims.
[0094] It is revealed that the present disclosure was carried out
with the support of the Ministry of Science, Technology and
Communication of the Republic of Korea (NRF-2018M3A9C8023568) and
the Ministry of Education of the Republic of Korea
(2019R1A6A11036849).
Sequence CWU 1
1
56118PRTArtificial Sequenceprotein transducing domain 1Ala Ala Asn
Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val218PRTArtificial Sequenceprotein transducing domain 2Arg Ala Asn
Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val318PRTArtificial Sequenceprotein transducing domain GK1-2 3Arg
Arg Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10
15Gly Val418PRTArtificial Sequenceprotein transducing domain GK1-3
4Arg Arg Arg Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5
10 15Gly Val518PRTArtificial Sequenceprotein transducing domain
GK1-4 5Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr
Val1 5 10 15Gly Arg618PRTArtificial Sequenceprotein transducing
domain GK1-5 6Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala
Asp Thr Val1 5 10 15Arg Arg718PRTArtificial Sequenceprotein
transducing domain GK1-6 7Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala
Glu Leu Ala Asp Thr Arg1 5 10 15Arg Arg818PRTArtificial
Sequenceprotein transducing domain GK1-7 8Ala Arg Asn Asp Leu Ala
Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val918PRTArtificial Sequenceprotein transducing domain GK1-8 9Ala
Ala Arg Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10
15Gly Val1018PRTArtificial Sequenceprotein transducing domain GK1-9
10Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1
5 10 15Arg Val1118PRTArtificial Sequenceprotein transducing domain
GK1-10 11Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp
Thr Arg1 5 10 15Gly Val1219PRTArtificial Sequenceprotein
transducing domain GK1-11 12Arg Ala Ala Asn Asp Leu Ala Glu Gly Leu
Ala Glu Leu Ala Asp Thr1 5 10 15Val Gly Val1320PRTArtificial
Sequenceprotein transducing domain GK1-12 13Arg Arg Ala Ala Asn Asp
Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp1 5 10 15Thr Val Gly Val
201421PRTArtificial Sequenceprotein transducing domain GK1-13 14Arg
Arg Arg Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala1 5 10
15Asp Thr Val Gly Val 201519PRTArtificial Sequenceprotein
transducing domain GK1-14 15Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala
Glu Leu Ala Asp Thr Val1 5 10 15Gly Val Arg1620PRTArtificial
Sequenceprotein transducing domain GK1-15 16Ala Ala Asn Asp Leu Ala
Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly Val Arg Arg
201721PRTArtificial Sequenceprotein transducing domain GK1-16 17Ala
Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10
15Gly Val Arg Arg Arg 201827PRTArtificial Sequenceprotein
transducing domain GK1-17 18Arg Lys Lys Arg Arg Gln Arg Arg Arg Ala
Ala Asn Asp Leu Ala Glu1 5 10 15Gly Leu Ala Glu Leu Ala Asp Thr Val
Gly Val 20 251927PRTArtificial Sequenceprotein transducing domain
GK1-18 19Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala Asp
Thr Val1 5 10 15Gly Val Arg Lys Lys Arg Arg Gln Arg Arg Arg 20
252025PRTArtificial Sequenceprotein transducing domain GK1-19 20Lys
Glu Thr Trp Trp Glu Thr Ala Ala Asn Asp Leu Ala Glu Gly Leu1 5 10
15Ala Glu Leu Ala Asp Thr Val Gly Val 20 252129PRTArtificial
Sequenceprotein transducing domain GK1-20 21Glu Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val Ala Ala Asn Asp Leu1 5 10 15Ala Glu Gly Leu Ala
Glu Leu Ala Asp Thr Val Gly Val 20 252225PRTArtificial
Sequenceprotein transducing domain GK1-21 22Ala Ala Asn Asp Leu Ala
Glu Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly Val Lys Glu Thr
Trp Trp Glu Thr 20 252329PRTArtificial Sequenceprotein transducing
domain GK1-22 23Ala Ala Asn Asp Leu Ala Glu Gly Leu Ala Glu Leu Ala
Asp Thr Val1 5 10 15Gly Val Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys
Val 20 252454DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1 24gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgta 542554DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-1
25cgcgcgaatg atcttgcgga aggtctggcg gaactcgcgg atacggtagg cgta
542654DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-2 26cgccgtaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgta 542754DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-3
27cgccgtcgcg atcttgcgga aggtctggcg gaactcgcgg atacggtagg cgta
542854DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-4 28gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg ccgc 542954DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-5
29gctgcgaatg atcttgcgga aggtctggcg gaactcgcgg atacggtacg ccgc
543054DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-6 30gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacgcgtcg ccgc 543154DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-7
31gctcgcaatg atcttgcgga aggtctggcg gaactcgcgg atacggtagg cgta
543254DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-8 32gctgcgcgcg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgta 543354DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-9
33gctgcgaatg atcttgcgga aggtctggcg gaactcgcgg atacggtacg cgta
543454DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-10 34gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacgcgcgg cgta 543557DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-11
35cgcgctgcga atgatcttgc ggaaggtctg gcggaactcg cggatacggt aggcgta
573660DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-12 36cgccgtgctg cgaatgatct tgcggaaggt
ctggcggaac tcgcggatac ggtaggcgta 603763DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-13
37cgccgtcgcg ctgcgaatga tcttgcggaa ggtctggcgg aactcgcgga tacggtaggc
60gta 633857DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-14 38gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgtacgc 573960DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-15
39gctgcgaatg atcttgcgga aggtctggcg gaactcgcgg atacggtagg cgtacgccgt
604063DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-16 40gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgtacgccgt 60cgc 634181DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-17
41aggaagaagc ggagacagcg acgaagagct gcgaatgatc ttgcggaagg tctggcggaa
60ctcgcggata cggtaggcgt a 814281DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-18
42gctgcgaatg atcttgcgga aggtctggcg gaactcgcgg atacggtagg cgtaaggaag
60aagcggagac agcgacgaag a 814375DNAArtificial
Sequencepolynucleotide encoding protein transducing domain GK1-19
43aaagaaacct ggtgggaaac cgctgcgaat gatcttgcgg aaggtctggc ggaactcgcg
60gatacggtag gcgta 754487DNAArtificial Sequencepolynucleotide
encoding protein transducing domain GK1-20 44gaatggtctc agccgaaaaa
aaaacgtaaa gtggctgcga atgatcttgc ggaaggtctg 60gcggaactcg cggatacggt
aggcgta 874575DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-21 45gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgtaaaagaa 60acctggtggg aaacc
754687DNAArtificial Sequencepolynucleotide encoding protein
transducing domain GK1-22 46gctgcgaatg atcttgcgga aggtctggcg
gaactcgcgg atacggtagg cgtagaatgg 60tctcagccga aaaaaaaacg taaagtg
874760DNAArtificial Sequencesense oligonucleotide 47tatggctgcg
aatgatcttg cggaaggtct ggcggaactc gcggatacgg taggcgtaca
604860DNAArtificial Sequenceantisense oligonucleotide 48tatgtacgcc
taccgtatcc gcgagttccg ccagaccttc cgcaagatca ttcgcagcca
604918PRTArtificial SequencepeptideVARIANT(1)..(1)Xaa is Ala or
ArgVARIANT(2)..(2)Xaa is Ala or ArgVARIANT(3)..(3)Xaa is Asn or
ArgVARIANT(16)..(16)Xaa is Val or ArgVARIANT(17)..(17)Xaa is Gly or
ArgVARIANT(18)..(18)Xaa is Val or Arg 49Xaa Xaa Xaa Asp Leu Ala Glu
Gly Leu Ala Glu Leu Ala Asp Thr Xaa1 5 10 15Xaa
Xaa5018PRTArtificial SequencePeptide 50Ala Ala Asn Asp Leu Ala Glu
Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val5118PRTArtificial SequencePeptide 51Ala Ala Asn Asp Leu Ala Glu
Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val5218PRTArtificial SeuquencePeptide 52Ala Ala Asn Asp Leu Ala Glu
Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly
Val5318PRTArtificial SequencePeptide 53Ala Ala Asn Asp Leu Ala Glu
Gly Leu Ala Glu Leu Ala Asp Thr Val1 5 10 15Gly Val549PRTArtificial
SequencePeptide 54Arg Lys Lys Arg Arg Gln Arg Arg Arg1
5557PRTArtificial SequencePeptide 55Lys Glu Thr Trp Trp Glu Thr1
55611PRTArtificial SequencePeptide 56Glu Trp Ser Gln Pro Lys Lys
Lys Arg Lys Val1 5 10
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